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Department of Physiology, Saga Medical School, Saga, Japan
Submitted 27 November 2007; accepted in final form 21 January 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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0.7 µM for half-maximal effect and were depressed by PLA2 inhibitor 4-bromophenacyl bromide or aristolochic acid. The melittin-induced enhancement of glycinergic transmission was depressed by lipoxygenase inhibitor nordihydroguaiaretic acid but not cyclooxygenase inhibitor indomethacin. These results indicate that the activation of PLA2 in the SG enhances GABAergic and glycinergic inhibitory transmission in SG neurons. The former action is mediated by glutamate-receptor activation and neuronal activity increase, possibly the facilitatory effect of PLA2 activation on excitatory transmission, whereas the latter action is due to PLA2 and subsequent lipoxygenase activation and is independent of extracellular Ca2+. It is suggested that PLA2 activation in the SG could enhance not only excitatory but also inhibitory transmission, resulting in the modulation of nociception. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Shimizu and Wolfe 1990). There is much evidence supporting an involvement of COX metabolites prostanoids in enhancing nociceptive transmission in the spinal dorsal horn (for review, see Samad et al. 2002; Svensson and Yaksh 2002; Vanegas and Schaible 2001). For instance, Dirig and Yaksh (1999) have demonstrated that peripheral tissue injury and inflammation increase the release of prostanoids such as prostaglandin E2 (PGE2), possibly through PLA2 activation, in the rat spinal cord. Baba et al. (2001) have reported that bath-applied PGE2 produces a depolarization in dorsal horn neurons of rat spinal cord slices. Alternatively, it is possible that metabolites of LOX are involved in nociceptive transmission because the metabolites directly activate TRPV1, a molecule that plays a role in producing nociception (Hwang et al. 2000).
Melittin, a major component of bee venom, is a 26 amino-acid peptide that is known to have a variety of bioactive actions including PLA2 activation and facilitation of synaptic transmission in the CNS (Aronica et al. 1992; Bernard et al. 1995; Phillis et al. 1999). We have recently reported that melittin reversibly enhances glutamatergic excitatory transmission in spinal dorsal horn substantia gelatinosa (SG) neurons (Yue et al. 2005), which play a crucial role in regulating nociceptive transmission to the CNS from the periphery (for review, see Willis and Coggeshall 1991). This action was due to PLA2 activation without an involvement of arachidonic acid metabolites produced by COX and LOX (Yue et al. 2005). The SG neurons receive not only excitatory transmission but also GABAergic and glycinergic inhibitory transmission (Willis and Coggeshall 1991). There is much evidence showing that inhibitory transmission in the spinal dorsal horn may contribute to nociceptive transmission. For example, Moore et al. (2002) have reported an inhibition of primary-afferent-evoked inhibitory transmission in SG neurons and a reduction in the level of spinal dorsal horn GABA-synthesizing enzyme expression in partial nerve injury rat models compared with naive rats. Local blockade or knock-down of the potassium-chloride exporter KCC2 in the spinal dorsal horn, something that results in shift in transmembrane chloride gradient and thus causes normally inhibitory anionic synaptic currents to be excitatory, markedly reduced nociceptive thresholds in rats (Coull et al. 2003). To our knowledge, it has not been examined yet at the cellular level how PLA2 activation itself affects inhibitory transmission in the spinal dorsal horn, although Ahmadi et al. (2002) have reported that PGE2 blocks glycinergic transmission in rat superficial dorsal horn neurons (for review, see Zeilhofer 2005). To know a role of PLA2 in modulating inhibitory transmission at the spinal cord level, we examined the effect of melittin on spontaneous inhibitory postsynaptic currents (sIPSCs) in SG neurons of adult rat spinal cord slice preparations by using the whole cell patch-clamp technique.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Slice preparation
Spinal cord slices from adult rats were prepared as described previously (Fujita and Kumamoto 2006; Liu et al. 2004). In brief, male adult Sprague-Dawley rats (6–8 wk old) were anesthetized with urethan (1.2 g/kg ip), and then a laminectomy was performed to extract a lumbosacral spinal cord enlargement (L1–S3). The spinal cord was placed in preoxygenated Krebs solution at 1–3°C. After cutting all of ventral and dorsal roots, the pia-arachnoid membrane was removed. The spinal cord was mounted on a vibrating microslicer and then a 600-µm thick transverse slice was cut. The slice was placed on a nylon mesh in the recording chamber (volume: 0.5 ml) and then perfused at a rate of 10–15 ml/min with Krebs solution bubbled with 95% O2-5% CO2, and maintained at 36 ± 1°C. The Krebs solution contained (in mM) 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, and 11 glucose (pH = 7.4 when saturated with the gas).
Whole cell voltage-clamp recordings
The SG was identified as a translucent band under a binocular microscope with light transmitted from below (Fujita and Kumamoto 2006; Liu et al. 2004; Nakatsuka et al. 1999). Blind whole cell voltage-clamp recordings from SG neurons were made at a holding potential (VH) of 0 mV where spontaneous glutamatergic excitatory postsynaptic currents (EPSCs) were invisible because the reversal potential for non-N-methyl-D-aspartate (non-NMDA) receptor channels involved in this production was close to 0 mV (Kohno et al. 1999; Yang et al. 2004) unless otherwise mentioned. Patch-pipettes were fabricated from thin-walled, fiber-filled capillary (1.5 mm OD) and contained the following solution (in mM): 110 Cs2SO4, 0.5 CaCl2, 2 MgCl2, 5 EGTA, 5 HEPES, 5 Mg-ATP, and 5 tetraethylammonium (TEA)-Cl (pH = 7.2). In some experiments, 110 mM-Cs2SO4 in the patch-pipette solution was replaced by 80 mM-Cs2SO4 and 30 mM-CsCl, and the tonicity of this solution was adjusted by adding sucrose, which enabled to record sIPSCs having a considerable amplitude at –70 mV. Signals were acquired using an EPC-7 or Axopatch 200B amplifier. Currents obtained in the voltage-clamp mode were low-pass-filtered at 3 or 5 kHz and digitized at 333 or 500 kHz with an A/D converter (Digidata 1200 or 1322A). The data were stored and analyzed with a personal computer using pCLAMP v 9.2 software. The program (AxoGraph 4.0) used for analyzing sIPSCs detects spontaneous events if the difference between the baseline and a following current value exceeds a given threshold of 5 pA and separating valleys are <50% of adjacent peaks.
Application of drugs
Drugs were applied by perfusing a solution containing drugs of a known concentration without an alteration in the perfusion rate and temperature. The solution in the recording chamber having a volume of 0.5 ml was completely replaced within 15 s. The drugs used were tetrodotoxin (TTX; Wako, Osaka, Japan), melittin purified from bee venom, 4-bromophenacyl bromide (4-BPB), indomethacin (INDO), nordihydroguaiaretic acid (NDGA), strychnine nitrate, bicuculline methiodide, PLA2 from bee venom, aristolochic acid, DL-2-amino-5-phosphonovaleric acid (APV; Sigma, St. Louis, MO), leukotriene B4 (LTB4; Cayman Chemicals, Ann Arbor, MI), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Tocris Cookson, Bristol, UK). These drugs (except for melittin, PLA2, TTX, bicuculline, strychnine and APV where distilled water was used as solvent) were first dissolved in dimethyl sulfoxide (DMSO) at 1000 (500 for INDO and NDGA) times the concentration to be used and then stored at –20°C. The stock solution was diluted to the desired concentration in Krebs solution immediately before use. The tonicity of nominally Ca2+-free, high-Mg2+ (5 mM) Krebs solution was adjusted by lowering Na+ concentration of Krebs solution. When melittin (1 µM) was repeatedly superfused in the same spinal cord slice, time intervals between the application were >1 h.
Statistical analysis
Numerical data are presented as the means ± SE. Statistical significance was determined as P < 0.05 using either Student's t-test (unless otherwise noted) or Kolmogorov-Smirnov test. In all cases, n refers to the number of neurons studied.
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RESULTS |
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). The effects of melittin on the inhibitory transmission were examined in a total of 238 SG neurons. Effect of melittin on inhibitory synaptic transmission in SG neurons
Melittin (1 µM) superfused for 3 min increased the frequency and amplitude of sIPSCs recorded at 0 mV in a reversible manner as seen in Fig. 1A. These actions were visible 2 min after the beginning of melittin superfusion. Because melittin is known to produce a deteriorative effect on cell membranes (Fletcher and Jiang 1993), this peptide was not superfused for >3 min at one time. When measured 3 min after the beginning of its superfusion, sIPSC frequency and amplitude were, respectively, 549 ± 134% (n = 7; P < 0.05) and 222 ± 21% (n = 7; P < 0.05) of control (1.8 ± 0.3 Hz and 14.2 ± 2.2 pA). Two types of GABAergic and glycinergic sIPSCs were encountered in SG neurons as reported previously (Kohno et al. 1999; Yang et al. 2004). A non-NMDA receptor antagonist CNQX (10 µM) did not affect GABAergic sIPSCs [amplitude and frequency: 97 ± 2 and 103 ± 9%, respectively, of control (9.8 ± 0.9 pA and 2.1 ± 0.3 Hz); n = 4] and glycinergic sIPSCs [amplitude and frequency: 101 ± 3 and 106 ± 10%, respectively, of control (12.0 ± 2.3 pA and 2.3 ± 0.3 Hz); n = 4], indicating no contamination of spontaneous EPSCs in sIPSCs. As seen in Fig. 1B, GABAergic sIPSCs, which were observed in the presence of a glycine-receptor antagonist strychnine (1 µM), were enhanced in frequency and amplitude by melittin (1 µM). Table 1 gives GABAergic sIPSC frequency and amplitude, measured 3 min after the beginning of melittin superfusion, relative to control, which are obtained from 32 neurons. In the presence of a GABAA-receptor antagonist bicuculline (10 µM), glycinergic sIPSCs, which were shorter in duration than GABAergic ones (compare sIPSC traces in expanded scale in time in Fig. 1, B and C), could be recorded. As seen for GABAergic sIPSCs, melittin (1 µM) increased glycinergic sIPSC frequency and amplitude (Fig. 1C). Table 1 shows the effects of melittin on glycinergic sIPSC frequency and amplitude, which are obtained from 27 neurons.
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Melittin enhances GABAergic but not glycinergic transmission by facilitating glutamatergic transmission
To know whether the sIPSC frequency and amplitude increases produced by melittin are accompanied by an increase in neuronal activities, we next examined how the melittin-induced enhancement is affected by TTX (0.5 µM). Figures 2A and 3A demonstrate the effects of melittin (1 µM) on GABAergic and glycinergic transmission, respectively, in Krebs solution containing TTX. Melittin did not affect GABAergic miniature IPSC (mIPSC, i.e., sIPSC in the presence of TTX) frequency and amplitude while increasing glycinergic mIPSC ones. Figures 2C and 3C and Table 1 summarize the effects of melittin on GABAergic and glycinergic mIPSC frequency and amplitude, which are examined in 19 and 9 neurons, respectively. The GABAergic mIPSC frequency and amplitude was unaffected by melittin (Fig. 2C and Table 1). On the contrary, melittin increased glycinergic mIPSC frequency and amplitude; the extents of the increases were not significantly different from those for glycinergic sIPSCs (Fig. 3C).
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), which results in an increase in neuronal activities. To examine this possibility, next, we examined the effect of melittin (1 µM) on GABAergic transmission in Krebs solution containing CNQX (10 µM; data not shown). CNQX partially depressed the melittin-induced increases in GABAergic sIPSC frequency and amplitude; GABAergic sIPSC frequency and amplitude, measured around 3 min after the beginning of melittin superfusion, relative to control, which are obtained from ten neurons, are shown in Table 1. Adding APV (50 µM) to CNQX-containing Krebs solution resulted in blocking the facilitatory effect of melittin (1 µM) on GABAergic transmission, as seen in Fig. 2B; data obtained from 12 neurons are given in Fig. 2C and Table 1. Unlike GABAergic sIPSCs, glycinergic sIPSCs were enhanced in frequency and amplitude by melittin (1 µM) in Krebs solution containing only CNQX (10 µM; n = 7) or both CNQX (10 µM) and APV (50 µM; n = 4; see Fig. 3B and Table 1). There was not a significant difference in glycinergic sIPSC frequency and amplitude increases produced by melittin between the absence and presence of glutamate-receptor antagonists (Fig. 3C).
If the GABAergic transmission enhancement produced by melittin is due to its effect on excitatory transmission, this enhancement is expected to be reduced in extent in Ca2+-free Krebs solution because of the lack of the facilitatory effect of melittin (1 µM) on excitatory transmission in Ca2+-free Krebs solution (see Yue et al. 2005). To confirm this idea, we examined the effect of melittin (1 µM) on GABAergic transmission in a nominally Ca2+-free, high-Mg2+ (5 mM) Krebs solution. Superfusing Ca2+-free Krebs solution reduced the frequency but not amplitude of GABAergic sIPSC [they were, respectively, 84 ± 4% (n = 13) and 95 ± 2% (n = 13) of control (3.1 ± 0.3 Hz and 10.5 ± 0.4 pA)] under the condition of which melittin (1 µM) did not affect GABAergic sIPSCs (Fig. 4Aa). Data obtained from 13 neurons are given in Table 1 and Fig. 4Ab. On the contrary, glycinergic sIPSC frequency and amplitude were increased by melittin (1 µM) in Ca2+-free solution (Fig. 4Ba) where the frequency but not amplitude of glycinergic sIPSC was reduced [they were, respectively, 75 ± 8% (n = 13) and 92 ± 6% (n = 13) of control (3.4 ± 0.8 Hz and 13.7 ± 1.3 pA)]. Table 1 gives data obtained from eight neurons; there was not a significant difference in the extents of glycinergic sIPSC frequency and amplitude increases produced by melittin between the presence and absence of extracellular Ca2+ (Fig. 4Bb).
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have reported similar observations in mouse SG neurons and attributed the inward current to a vesicular release of GABA from nerve terminals. Characterization of the glycinergic transmission enhancement produced by melittin
Because no effects of TTX, glutamate-receptor antagonists and nominally Ca2+-free solution on melittin-induced glycinergic transmission enhancement suggest a direct action of melittin on glycinergic transmission, we next characterized the glycinergic transmission enhancement produced by melittin. Figure 5, A and B demonstrates the time course of a change in glycinergic sIPSC frequency and amplitude following superfusion of melittin (1 µM). Both of its frequency and amplitude increased with a delay of 1–2 min after the beginning of melittin superfusion, and these increases subsided within 10 min after washout. Figure 5C demonstrates cumulative distributions of the inter-event interval and amplitude of glycinergic sIPSC in the control and under the effect of melittin. Melittin significantly increased the proportion of glycinergic sIPSCs having a shorter inter-event interval and of those having a larger amplitude; this effect was confirmed in three other neurons. Figure 5D demonstrates an average of glycinergic sIPSC traces in the absence and presence of melittin in the same neuron as that of Fig. 5, A–C, and their superimposition where control glycinergic sIPSC trace is scaled in amplitude to that under the effect of melittin. As judged from this superimposition, the glycinergic sIPSC amplitude increase produced by melittin was not accompanied by a change in its decay phase. When examined in four neurons, the half-decay times of glycinergic sIPSC in the absence and presence of melittin were 3.7 ± 0.3 and 3.8 ± 0.3 ms, respectively; they were not significantly different (P > 0.05). Figure 6 demonstrates the effect of a repeated application of melittin (1 µM) on glycinergic transmission in the same neuron. When melittin was once again applied 30 min after its washout, the effects of the second application of melittin on glycinergic sIPSC frequency and amplitude (Fig. 6B) were much smaller in extent than those of the first application of melittin (Fig. 6A). Figure 6C shows a comparison of relative frequency and amplitude of glycinergic sIPSC under the action of melittin to those before its superfusion between the first and second applications, which was obtained from six neurons. When examined in different SG neurons in the same slice preparation >1 h after washout of melittin (1 µM), the facilitatory effect on glycinergic transmission of the second application of melittin was not distinct from that of its first application over a variation of sIPSC frequency and amplitude increases among neurons (data not shown).
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Because melittin is known to have effects other than PLA2 activation (for review, see Fletcher and Jiang 1993), first we examined whether the glycinergic transmission enhancements produced by melittin are really mediated by PLA2 by using a PLA2 inhibitor 4-BPB (Mayer and Marshall 1993). The pretreatment for 4 min with 4-BPB [10 µM, a concentration used previously by us to examine an involvement of PLA2 in the melittin-induced enhancement of glutamatergic transmission (Yue et al. 2005)] partially inhibited the effects of melittin (1 µM) on glycinergic sIPSC frequency and amplitude; the frequency and amplitude around 3 min after the beginning of melittin superfusion, relative to control, which was obtained from eight cells, is given in Table 1. This concentration of 4-BPB may not have been enough to inhibit the melittin-induced enhancement of glycinergic transmission because this enhancement is much larger in extent than that of glutamatergic transmission (see Yue et al. 2005). When examined in the presence of 4-BPB at a higher concentration of 50 µM, melittin (1 µM) did not affect glycinergic transmission as shown in Fig. 8A. Data obtained from four neurons are summarized in Table 1 and Fig. 8C. 4-BPB itself at 50 µM did not affect glycinergic sIPSC frequency and amplitude [99 ± 3% (n = 3; P > 0.05) and 94 ± 8% (n = 3; P > 0.05), respectively, of control] around 4 min after the beginning of its superfusion (data not shown). Figure 8, B and C, demonstrates how another PLA2 inhibitor aristolochic acid (Vishwanath et al. 1987) affects the melittin-induced enhancement of glycinergic transmission. As seen in Fig. 8B, melittin (1 µM) did not increase glycinergic sIPSC frequency and amplitude under the condition of the pretreatment with aristolochic acid (100 µM, a concentration enough to reduce melittin-induced increase in ATP-sensitive K+ channel activity in rat pituitary GH3 cells; Wu et al. 2000) for 4 min. Data obtained from six neurons are given in Table 1 and Fig. 8C. Aristolochic acid (100 µM) itself did not affect glycinergic transmission; the frequency and amplitude of the sIPSC were, respectively, 101 ± 5% (n = 6; P > 0.05) and 95 ± 3% (n = 6; P > 0.05) of control around 4 min after the beginning of its superfusion (data not shown).
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) and increases AMPA receptor affinity in the rat SG (Cruickshank and Henley 1994) and telencephalon (Massicotte and Baudry 1990), bee venom PLA2 itself at 0.1 or 4 unit/ml did not affect glycinergic sIPSC frequency and amplitude [0.1 unit/ml: 100 ± 5% (n = 6; P > 0.05) and 97 ± 2% (n = 6; P > 0.05), respectively, of control (3.2 ± 0.4 Hz and 14.6 ± 0.8 pA); 4 unit/ml: 99 ± 3% (n = 4; P > 0.05) and 96 ± 4% (n = 4; P > 0.05), respectively, of control (2.8 ± 0.5 Hz and 14.1 ± 0.6 pA)] 3 min after the beginning of its superfusion (data not shown). Thus it is unlikely that PLA2, which may have been present in Krebs solution containing melittin as used in the present study, enhanced glycinergic transmission in SG neurons (see Metz 1986).
To know whether the cascade of arachidonic acid following PLA2 activation is involved in the melittin effect, we next examined the effect of melittin (1 µM) on glycinergic transmission under the condition of the pretreatment with a COX inhibitor INDO or a LOX inhibitor NDGA [each at 100 µM for 4 min as done in our previous study (Yue et al. 2005)]. In the presence of INDO, melittin increased glycinergic sIPSC frequency and amplitude, as seen in Fig. 9A. Data obtained from seven neurons are given in Table 1. There was not a significant difference in the extents of glycinergic sIPSC frequency and amplitude increases produced by melittin between the absence and presence of INDO (Fig. 9C). On the other hand, the effect of melittin on glycinergic transmission was not seen in the presence of NDGA (Fig. 9B). Table 1 shows data obtained from nine neurons; glycinergic sIPSC frequency and amplitude 3 min after the beginning of melittin superfusion in the presence of NDGA, relative to control were not significantly different from 100% (Fig. 9C). INDO and NDGA (each 100 µM) by themselves did not affect glycinergic sIPSC frequency and amplitude [INDO: 104 ± 11% (n = 7; P > 0.05) and 101 ± 3% (n = 7; P > 0.05), respectively, of control; NDGA: 96 ± 7% (n = 6; P > 0.05) and 106 ± 5% (n = 6; P > 0.05), respectively, of control]. DMSO (0.2%) contained in the INDO or NDGA solution by itself did not affect glycinergic transmission; the frequency and amplitude of the sIPSC were, respectively, 101 ± 8% (n = 3; P > 0.05) and 100 ± 5% (n = 3; P > 0.05) of control (2.7 ± 0.6 Hz and 14.1 ± 1.5 pA) around 4 min after the beginning of its superfusion.
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) and increases intracellular Ca2+ concentrations in dorsal root ganglion neurons (Andoh and Kuraishi 2005), LTB4 was tested as a candidate of endogenous substances involved in the enhancement of glycinergic transmission as a result of PLA2 activation. LTB4 at 0.1 µM (a concentration enough to activate LTB4 receptors in vascular smooth muscle cells) (Bäck et al. 2005) did not affect glycinergic transmission [frequency and amplitude of the sIPSC: 99 ± 8% (n = 3; P > 0.05) and 100 ± 2% (n = 3; P > 0.05), respectively, of control (2.6 ± 0.7 Hz and 12.5 ± 1.4 pA) 3 min after the beginning of LTB4 superfusion; data not shown]. In SG neurons where melittin (1 µM) increased glycinergic sIPSC frequency and amplitude [329 ± 21% (n = 3; P < 0.05) and 201 ± 39% (n = 3; P < 0.05), respectively, of control (2.6 ± 0.5 Hz and 14.4 ± 1.9 pA) around 3 min after the beginning of melittin superfusion], LTB4 superfused at 0.5 µM before the application of melittin did not affect them [frequency and amplitude: 107 ± 8% (n = 3; P > 0.05) and 98 ± 12% (n = 3; P > 0.05), respectively, of control (2.4 ± 0.3 Hz and 14.8 ± 1.9 pA) 3 min after the beginning of LTB4 superfusion; data not shown]. |
DISCUSSION |
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); the enhancement by melittin of glutamatergic spontaneous excitatory transmission in SG neurons has been previously reported by Yue et al. (2005). The glycinergic transmission enhancement produced by melittin was resistant to TTX and glutamate-receptor antagonists (CNQX and APV), indicating a direct action of melittin on glycinergic transmission. On the other hand, the GABAergic transmission enhancement produced by melittin was not seen in the presence of TTX or glutamate-receptor antagonists and in a Ca2+-free Krebs solution, indicating an involvement of the facilitatory effect of melittin on excitatory transmission and of an increase in neuronal activities in the SG. Enhancement by melittin of glycinergic inhibitory transmission in SG neurons
The melittin-induced enhancement of glycinergic transmission is both pre- and postsynaptic in origin because melittin increases the proportion of glycinergic sIPSCs having a shorter inter-event interval and of those having a larger amplitude. Consistent with this presynaptic action, Phillis et al. (1999) have reported that melittin enhances the release of glycine from the rat cerebral cortex in vivo. EC50 values for the pre- and postsynaptic actions in SG neurons were, respectively, 0.73 and 0.69 µM, values comparable to that (1.1 µM) for the presynaptic action at glutamatergic synapses in the SG (Yue et al. 2005). These values were larger by about sevenfold than that (0.11 µM) in enhancing aspartate release from cultured neurons (Aronica et al. 1992) while being smaller by about fivefold than that (3.5 µM) in increasing AMPA receptor affinity (Bernard et al. 1995). The glycinergic sIPSC amplitude increase produced by melittin is not accompanied by a change in the half-decay time of the sIPSC. This effect may be due to a change not in the channel kinetics of the glycine receptor but in its affinity for glycine because the decay phase of glycinergic synaptic current in spinal cord neurons is thought to be produced by a random closure of individual glycine-receptor channels (see Takahashi and Momiyama 1991). With respect to this postsynaptic effect, a rise in intracellular Ca2+ in postsynaptic neurons appears not to be involved in the glycinergic sIPSC amplitude increase produced by melittin because the patch-pipette solution used in the present study contained 5 mM EGTA, which may have chelated intracellular Ca2+ being involved in increasing an apparent affinity of the glycine receptor for glycine as a result of melittin actions (see Fucile et al. 2000).
Melittin did not significantly affect glycinergic transmission in the presence of 4-BPB or aristolochic acid, suggesting an involvement of PLA2 activation. In support of this idea, the spinal cord contains secreted and cytosolic PLA2 (Samad et al. 2001; for review, see Svensson and Yaksh 2002). Although it is known that arachidonic acid produced as a result of PLA2 activation is metabolized by various enzymes including COX and LOX (Shimizu and Wolfe 1990), the disappearance of this melittin effect in the presence of NDGA but not INDO suggests an involvement of the metabolites of LOX but not COX. The lack of the effect of INDO on the melittin-induced increase in glycinergic sIPSC amplitude suggests that an inhibitory effect of PGE2 on glycine-receptor responses in rat superficial dorsal horn neurons (Ahmadi et al. 2002; for review, see Zeilhofer 2005) may have not contributed to the effect of PLA2 activation on glycinergic sIPSC amplitudes in the present study, probably because of an insufficient expression of COX and PGE synthase such as microsomal PGE2 synthase-1 (Guay et al. 2004), both of which are required for spinal PGE2 production (for review, see Shimizu and Wolfe 1990). Consistent with an involvement of such a metabolite system, a repeated application of melittin at 30-min interval had almost no effects on glycinergic transmission. This was so even when glycinergic sIPSC frequency and amplitude recovered to those before the application of melittin. An intracellular system leading to the enhancement of glycinergic transmission may exhibit a desensitization. As the melittin-induced enhancement of glycinergic transmission persists in a Ca2+-free Krebs solution, this effect is independent of extracellular Ca2+. Because one of LOX metabolites, LTB4, does not affect glycinergic transmission in SG neurons, other LOX metabolites than LTB4 appear to be involved in the glycinergic transmission enhancement produced by PLA2 activation. Intracellular mechanisms for glycinergic transmission enhancement following PLA2 activation remain to be examined further.
Enhancement by melittin of GABAergic inhibitory transmission in SG neurons
The melittin-induced enhancement of GABAergic transmission may be mediated by neuromodulators released from a neuron as a result of excitatory transmission enhancement caused by PLA2 activation and subsequent increase in neuronal activities because the former action is resistant to TTX (see Yue et al. 2005), and thus the enhancement is expected to produce action potentials. In support of this idea, it is known that many kinds of neuromodulators enhance GABAergic inhibitory transmission in SG neurons. For instance, acetylcholine enhances inhibitory transmission in SG neurons by activating nicotinic and muscarinic acetylcholine receptors (Baba et al. 1998; Takeda et al. 2003). Norepinephrine increases the amplitude and frequency of GABAergic sIPSC in SG neurons (Baba et al. 2000b). It remains to be examined what kinds of neuromodulators are involved in the GABAergic transmission enhancement produced by PLA2 activation.
It is of interest to note that glycinergic but not GABAergic transmission is presynaptically affected by PLA2 activation. This suggests that GABA and glycine may be released from a different nerve terminal containing either GABA or glycine as different from the case in the lamina I where they are thought to be co-released from a single nerve terminal (Chéry and de Koninck 1999). This may be consistent with the observations that some presynaptic terminals in the SG contain GABA without glycine (Spike and Todd 1992) and that GABAergic transmission is affected by norepinephrine in a distinct manner from that of glycinergic transmission in SG neurons (Baba et al. 2000a). The presence of a tonic GABA but not glycine current may be in part due to the fact that GABA and glycine are released from nerve terminals different from each other.
Physiological significance of PLA2 activation in the SG
The present study together with our previous one (Yue et al. 2005) revealed that PLA2 activation in the SG enhances glycinergic inhibitory and glutamatergic excitatory transmission, the latter of which actions leads to the enhancement of GABAergic inhibitory transmission. Analgesics such as opioids at the spinal dorsal horn level generally hyperpolarize membranes and inhibit glutamatergic transmission in SG neurons (Kohno et al. 1999; Fujita and Kumamoto 2006; for review, see Fürst 1999), both of which reduce the excitability of the neurons, an effect of the enhancement of glycinergic transmission. Neuromodulators such as acetylcholine and norepinephrine, which enhance GABAergic transmission in SG neurons, act as analgesics when administered intrathecally (Abram and O'Connor 1995; Abram and Winne 1995; Howe et al. 1983; Khan et al. 1998, 2001; Reddy et al. 1980). Thus PLA2 activation in the SG may inhibit nociceptive transmission from the periphery in a complex manner. Although Young et al. (1995) have suggested that PLA2 may be involved in a sustained increased activity of spinal dorsal horn neurons in response to a repeated application of mustard oil to the periphery, PLA2 activation would enhance not only excitatory but also inhibitory transmission in spinal dorsal horn neurons. Thus inflammation activates PLA2 and then induces pain sensitization (Dirig and Yaksh 1999), while PLA2 activation should enhance glycinergic transmission in SG neurons, an anti-hyperalgesic effect as shown in the present study. Taken into consideration that the glycinergic transmission enhancement is mediated by LOX but not COX metabolites, COX inhibitors given during inflammatory hyperalgesia (for instance, see Zhang et al. 1997) might shift the metabolism of arachidonic acid from COX to LOX metabolites, which could then contribute to the analgesic action of the COX inhibitors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. Kumamoto, Dept. of Physiology, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan (E-mail: kumamote{at}cc.saga-u.ac.jp)
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REFERENCES |
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